Project Summary Expansions of simple DNA repeats are implicated in more than thirty hereditary neurological and neurodegenerative disorders in humans. Hundreds of copies of the causative repeat can be added in just a few intergenerational transmissions. Thus, understanding the mechanisms responsible for large-scale repeat expansions is extremely important and has broad biomedical implications. My lab was the first to show that expandable DNA repeats stall replication fork progression in every experimental system studied, including bacteria, yeast and mammalian cells. This led us to propose that repeats can be added while the replication fork escapes from a ?repetitive trap?. Early models of repeat expansion involved slippage of repetitive DNA strands, which is normally small-scale, during DNA replication. Based on the size of expansions observed in humans, we believe that a distinct mechanism could cause large jumps in the repeat?s size. To substantiate this idea, we developed an experimental system for large-scale repeat expansions in a model organism, S. cerevisiae. This system uncovered features of repeat expansions similar to that observed in human pedigrees. The rate of expansions increased exponentially with their lengths. Repeat expansions become evident, when the length of a repeat exceeds the Okazaki fragment size, which is close to the repeat expansion threshold in humans. The majority of genes involved in repeat expansions appear to encode proteins of the replication or post- replication repair machineries. These observations led us to outline two pathways for large-scale repeat expansions based on either template-switching during DNA replication, or break-induced replication. Capitalizing on these achievements, we plan to move our research in three new directions. First, we are developing a novel experimental strategy to analyze repeat instability in non-dividing, chronologically aging yeast cells. Repeat expansions are known to occur in post-mitotic tissues, such as the brain, and they are believed to contribute to disease pathogenesis. Thus, understanding the genetic controls and mechanisms of repeat expansions in non-dividing cells is invaluable for understanding the pathobiology of these diseases. Second, we are working on establishing a genetically tractable system to analyze the mechanisms of large-scale repeat expansions in cultured mammalian cells. We will then look at the effect of candidate genes, which were identified in our yeast screens, on repeat expansions in mammalian cells using siRNA gene knockdown. Finally, while the length of an expandable repeat is the key factor determining disease inheritance, recent clinical genetics data point to the existence of trans-modifiers that can affect the likelihood of repeat expansions and disease progression. We will, therefore, identify trans- modifiers of repeat expansion at the genome-wide level in our yeast experimental system. Identification of such trans-modifiers is potentially very important for prognostic purposes and genetic counseling.